Project Summary/Abstract Rotaviruses (RVs) are the single most important cause of severe diarrhea in infants and young children worldwide. RV replicates predominantly in mature small intestinal epithelial cells. The young of many mammalian species are naturally infected with host-specific homologous RV that causes severe diarrheal disease virtually exclusively in that species. Replication of heterologous RV in the intestine is inefficient and this `species barrier' has been exploited to develop several licensed and candidate human RV vaccines. The mechanism for attenuated replication of these vaccines in humans is poorly understood due to lack of proper human models. Recent studies in a murine model of RV infection using both homologous murine RV and heterologous non-murine RV strains demonstrate that the failure to inhibit innate immunity by heterologous RV and restriction of target cell entry on a species-specific basis are the two critical determinants of host-specific replication in mice. The murine RV gene products most important in circumventing replication restriction are NSP1 and VP4. The mechanistic basis of modulating host-specific replication restriction involves suppression of the type I, II, and III interferon (IFN) responses via inhibition of IRF-3, NF-?B, and STAT1 signaling by NSP1 and modulation of viral binding to target epithelial cells mediated by VP4. Previous studies have compared murine and non-murine RV infection in vivo in suckling mice and/or murine cell culture but there is currently no information available to indicate whether or not host-range restriction in humans is mediated by identical, similar, or distinct mechanisms. In this proposal we will take advantage of a new, highly tractable human small intestinal organoid system that recapitulates the ability of human intestine to support vigorous replication of human origin RV strains but substantially restricts non-human origin RV strain replication. We will use this system and associated powerful single cell analytic tools to identify the genetic and mechanistic origins of RV replication restriction in people. Our work will expand our understanding of RV pathogenesis, will enhance our ability to design more effective third-generation RV vaccines and will increase our basic understanding of innate immunity in the human intestine. The Aims of this proposal are to: 1) Determine the genetic basis of host-range restriction of non-human origin RV strains in primary human intestinal epithelial cells. 2) Define the mechanistic basis of host-range restriction of non-human origin RV in the human gut.